Polymerase Chain Reaction Products Research Articles

The first molecular isolation of Halicephalobus gingivalis from horses in Iran.

Parasitic infections pose significant threats to humans' and animals' well-being worldwide. Among these parasites, Halicephalobus spp., a genus of nematodes, has gained attention due to its ability to cause severe infections in various animal species, including horses. This study aimed to determine the prevalence of Halicephalobus spp., specifically focusing on Halicephalobus gingivalis in horses. In July 2022, a cross-sectional study was conducted in northern Iran to determine the prevalence of Halicephalobus spp. Using standard coprological techniques, 141 fecal samples from randomly selected horses were analyzed for GI helminth eggs. The Halicephalobus spp. eggs present in faeces were identified by molecular methods. Polymerase Chain Reaction (PCR) was used to amplify the partial 5' variable region (~ 390 base pairs) of 18S DNA using SSUA_F and SSU22_R primers. Furthermore, the PCR products obtained were sequenced, and phylogenetic analysis was performed using available sequences from GenBank. Microscopic examination of 141 fresh faecal samples revealed 5 fecal samples were infected with small ellipsoidal nematode eggs ranging between 40 and 50 × 50-60μm. This study's PCR amplicons showed ~ 390bp bands on 2.0% agarose gel. A partial sequence of 18S DNA (363bp) was obtained herein (GenBank accession no. OQ843456). Overall, using molecular tools represents a significant step forward in diagnosing and managing the Halicephalobus gingivalis infections in horses.

Molecular characterization of toll-like receptor 2 gene in Bikaneri camels (Camelus dromedarius)

Toll-like receptors (TLRs) are an important family of receptors constituting the first line of defense system. Tolllike receptors play crucial roles in the innate immune system by recognizing pathogen-associated molecular patterns derived from various microbes. Present investigation was carried out to characterize the coding sequence of TLR2 gene in Indian dromedary camels. Blood was collected from five Bikaneri camels and genomic DNA was isolated. Eight pairs of overlapping primers were designed for amplification of TLR2 gene coding region. All the fragments were successfully amplified using Polymerase chain reaction (PCR). The PCR products were purified and subjected to Sanger’s sequencing. TLR2 gene fragment of 3213 bp consisting of 2287 bp of coding sequence of exon 2 and 926 bp of 3’untranslated region (UTR) was successfully amplified. The sequence generated was submitted to the NCBI with accession number MW557315.1. The encoded amino acids codes for 12 leucine rich repeats (LRR) (Accession no. UBN09134.1). At the nucleotide level, the Bikaneri camels’ TLR2 gene showed highest similarity with camelids (99.30 to 99.59%) followed by vicugna pacos (97.54%). The nucleotide sequence similarity was lowest with Homo sapiens (82.37%). The amino acid sequence based phylogenetic tree revealed close relationship between camelid species followed by pig (86.2%), whereas human, gorilla, horse and donkey were distantly placed on the tree.

A novel SNP (c.1311C>T) on heat shock protein 70 (HSP70) gene of Kacang goat in Indonesia

Heat shock proteins (HSPs) associated with stress reactions play an important role in cell survival by activating numerous regulatory proteins and inducing black apoptosis. This study aimed to identify the potential SNPs of HSP70 gene in Kacang goats in Indonesia. Forty-three Kacang goats were selected from Sidoarjo and Tulungagung districts. The DNA isolated from blood samples was successfully amplified using the polymerase chain reaction (PCR) method with a pair of primers. The PCR products were sequenced in the coding region. The sequences were successfully aligned to determine the potential SNPs. A novel SNP (c.1311C>T) was found in this study. This SNP was categorized as a synonymous mutation. The insertion-deletion (in-del) mutation was also observed at the 1151stand 1161st nucleotide positions. Based on these mutations, four haplotypes were constructed where haplotype 1 had the highest frequency in Sidoarjo. Indonesian goats had no close relation with Iraqi goats according to Neighbor-Joing with Kimura’s 2-parameter approach. Haplotypes 1 and 2 in Indonesian goats had three different bases with all haplotypes in Iraqi goats in Median Joining Network. The study concluded that a novel SNP of HSP70 gene was identified in Kacang goats.

Molecular characterization of toll-like receptor 2 gene in Bikaneri camels (Camelus dromedarius)

Toll-like receptors (TLRs) are an important family of receptors constituting the first line of defense system. Tolllike receptors play crucial roles in the innate immune system by recognizing pathogen-associated molecular patterns derived from various microbes. Present investigation was carried out to characterize the coding sequence of TLR2 gene in Indian dromedary camels. Blood was collected from five Bikaneri camels and genomic DNA was isolated. Eight pairs of overlapping primers were designed for amplification of TLR2 gene coding region. All the fragments were successfully amplified using Polymerase chain reaction (PCR). The PCR products were purified and subjected to Sanger’s sequencing. TLR2 gene fragment of 3213 bp consisting of 2287 bp of coding sequence of exon 2 and 926 bp of 3’untranslated region (UTR) was successfully amplified. The sequence generated was submitted to the NCBI with accession number MW557315.1. The encoded amino acids codes for 12 leucine rich repeats (LRR) (Accession no. UBN09134.1). At the nucleotide level, the Bikaneri camels’ TLR2 gene showed highest similarity with camelids (99.30 to 99.59%) followed by vicugna pacos (97.54%). The nucleotide sequence similarity was lowest with Homo sapiens (82.37%). The amino acid sequence based phylogenetic tree revealed close relationship between camelid species followed by pig (86.2%), whereas human, gorilla, horse and donkey were distantly placed on the tree.

A novel SNP (c.1311C>T) on heat shock protein 70 (HSP70) gene of Kacang goat in Indonesia

Heat shock proteins (HSPs) associated with stress reactions play an important role in cell survival by activating numerous regulatory proteins and inducing black apoptosis. This study aimed to identify the potential SNPs of HSP70 gene in Kacang goats in Indonesia. Forty-three Kacang goats were selected from Sidoarjo and Tulungagung districts. The DNA isolated from blood samples was successfully amplified using the polymerase chain reaction (PCR) method with a pair of primers. The PCR products were sequenced in the coding region. The sequences were successfully aligned to determine the potential SNPs. A novel SNP (c.1311C>T) was found in this study. This SNP was categorized as a synonymous mutation. The insertion-deletion (in-del) mutation was also observed at the 1151stand 1161st nucleotide positions. Based on these mutations, four haplotypes were constructed where haplotype 1 had the highest frequency in Sidoarjo. Indonesian goats had no close relation with Iraqi goats according to Neighbor-Joing with Kimura’s 2-parameter approach. Haplotypes 1 and 2 in Indonesian goats had three different bases with all haplotypes in Iraqi goats in Median Joining Network. The study concluded that a novel SNP of HSP70 gene was identified in Kacang goats.

Identification of the Membrane-Associated Transporter Protein (MATP) Gene Polymorphism as Candidate Mutation for Albinism in Japanese Quail

A pigmentation abnormality could be due to genetic mutation and trigger a disease related pigmentation deficiency such as albino which might be caused by mutation in membrane-associated transporter protein gene (MATP). The aim of this study was to identify polymorphism in the MATP exon 3 in Japanese quail. A total of nine Japanese quail consisting three brown plumage quail, three black plumage quail, and three albino quail were used in this study. Blood samples were used to extract the genomic Deoxyribonucleic acid (DNA) and used to amplify exon 3 regions of the MATP by polymerase chain reaction (PCR). The PCR products were then sequenced and analyzed. A total of 10 mutations were found, of which three mutations, g.4460G>A, g.4479G>A, and g.4514T>C, were within exon 3. These mutations were synonymous and non-synonymous. No specific mutation for albino was found in this study. In summary those mutations did not specifically determine albinism in Japanese quail.

Molecular Characterization of Paederus Spp (Coleoptera: Staphylinidae, Paederinae) the Agent of Human Linear Dermatitis in the Caspian Sea Coast, North of Iran.

A combined morphological and molecular survey was performed to determine the agent of human linear dermatitis Paederus Fabricius, 1775 (Coleoptera: Staphylinidae, Paederinae) species composition in Mazandaran Province in the Caspian Sea coast in northern Iran, where most of linear dermatitis cases of the country occurred. Altogether, 397 Paederus specimens were collected from May to August 2021 and classified using morphological characters and ITS2-rDNA sequence analysis. Morphological investigation revealed that all the specimens were Paederus fuscipes. ITS2 polymerase chain reaction (PCR) direct-sequences and the profiles of restriction fragment length polymorphism (RFLP) derived from digestion of PCR products by HinfI, HpaII, and SalI enzymes were identical confirming the morphological results, implying that all specimens belonged to a single taxon. Paederus fuscipes (Fabricius, 1775) is considered the dominant taxon and responsible for linear dermatitis in Mazandaran Province. To our knowledge, we have provided the first molecular typing of Paederus beetles at the species level, suggesting that ITS2-rDNA characterization is an alternative tool for species discrimination of Paederus spp.

Fast and highly sensitive screening of PCR products of severe acute respiratory syndrome coronavirus 2 by voltage‐programmed capillary gel electrophoretic separation

AbstractFast and highly sensitive detection has become critical to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). However, fast analysis tends to reduce the sensitivity and accuracy of such method. This study is to investigate a fast and highly sensitive screening method for detecting polymerase chain reaction (PCR) products of SARS‐CoV‐2 through voltage‐programmed capillary gel electrophoretic separation. By separating and detecting the PCR products of RNA‐dependent RNA polymerase (RdRp) and spike (S) genes, we have diagnosed coronavirus disease 2019 (COVID‐19) cases within 4 min using such method with high reliability and accuracy, indicating at least a 7.5‐fold faster than previous COVID‐19 diagnostic methods. The detection limits of RdRp and S genes fragments were 1.34–3.46 pM. The developed method can be used to analyze PCR products with specific DNA sizes as well as detect SARS‐CoV‐2 mutations without losing resolution. Furthermore, it can also be used for the fast analysis of DNA molecules related with various infectious diseases.

Application of polymerase chain reaction (PCR) methods in distinguishing pathogenic and saprophytic Leptospira from the traditional market environment in Denpasar City

Background: Leptospirosis disease is still a health problem in Indonesia. The number of leptospirosis cases and deaths in Indonesia tends to increase. Indirect transmission of Leptospira bacteria can occur through poor environmental sanitation polluted by rat urine has the potential to cause leptospirosis. Traditional markets are good places for rats to breed. The purpose of this study was to detect the presence of contamination by pathogenic and saprophytic Leptospira bacteria in the environment around traditional markets in Denpasar City by polymerase chain reaction (PCR) method using three specific primers. Methods: The sample is water from the market environment, taken at 19 location points from eight traditional markets in Denpasar City. After being homogenized, the sample water is filtered. Isolation of specific genes from samples by the PCR method was performed to differentiate pathogenic and saprophytic leptospira using three specific primers designed from the 16S rRNA gene. Starting from the DNA extraction stage, amplification by PCR, and detection of PCR product DNA by electrophoresis. Results: This study found that from 19 water sampling locations, 5/19 (26.3%) point locations found specific DNA genes for pathogenic Leptospira bacteria. 10/19 (52.6%) point locations found specific genes for saprophytic Leptospira bacteria DNA, and 4/19 (21.1%) point locations found no specific genes for pathogenic or saprophytic Leptospira bacteria. Conclusion: PCR examination using a combination of three primers could distinguish the presence of pathogenic Leptospira, saprophytic Leptospira, and the absence of pathogenic and saprophytic Leptospira simultaneously from traditional market environments in Denpasar City.

Structural Features and Genetic Diversity in Gag Gene of Rare HIV-1 Subtypes from the Democratic Republic of Congo.

Type-1 HIV (HIV-1) group M (HIV-1M) genetic diversity is highest in the Congo Basin where the epidemic ignited a century ago. HIV-1M has diversified into multiple subtypes, sub-subtypes, and circulating and unique recombinant forms (CRFs/URFs). An unanswered question is why some rare subtypes never reached epidemic levels despite their age. Several studies identified the role of HIV-1M accessory genes nef and vpu in virus adaptation to human hosts and subsequent spread. Other reports also pointed out the pivotal role of gag in transmissibility, virulence, and replication capacity. In this study we characterized the HIV-1 gag gene of 148 samples collected in different localities of the Democratic Republic of the Congo (DRC) between 1997 and 2013. We used nested polymerase chain reaction (PCR) to amplify the whole gag gene. PCR products were sequenced either by Sanger method or by next generation sequencing on Illumina MiSeq or iSeq100 platforms. Generated sequences were used for subsequent analyses using different bioinformatic tools. Phylogenetic analysis of the generated sequences revealed a high genetic diversity with up to 22 different subtypes, sub-subtypes, CRFs. Up to 15% (22/148) URFs were identified, in addition to rare subtypes such as H, J, and K. At least two amino acid motifs present in the gag gene have been shown to modulate HIV-1 replication, budding, and fitness: the P(T/S)AP and the LYPXnL motifs. Structural analysis revealed the presence of P(T/S)AP in all the 148 sequences with the majority (136/148) bearing the PTAP. Three samples presented a duplication of this motif. The LYPXnL motif was identified in 38 of 148 sequences. There was no clear link between the frequency of these motifs and HIV-1M subtypes. In summary, we confirmed a high genetic diversity of HIV-1M in the DRC. We observed the presence of amino acid motifs important for viral replication and budding even in some rare HIV-1 subtypes. Their impact on viral fitness needs be further evaluated by in vitro studies.

Identification of Candida auris by PCR and assessment of biofilm formation by crystal violet assay

IntroductionCandida auris is a notorious pathogen capable of forming biofilms on devices as well as host tissues, often culminating in infections. We evaluated characteristics of infections and the methods to diagnose C. auris over a period of three years in a tertiary care hospital. MethodsPatients admitted between 2018 and 2020, who had candidemia due to C. auris were included in the study. Identification was performed using HiCrome™ Candida Differential Agar, Vitek 2 (BioMérieux, Inc., Marcy-l’Etoile, France) and MALDI-TOF, Vitek-MS. Identification was confirmed by detection of rDNA region covering part of 5.8S, entire of ITS2, and part of 28S by polymerase chain reaction (PCR). Biofilm formation was assessed by crystal violet staining. ResultsPresence of central line and broad spectrum antimicrobials were noted in all patients whereas total parenteral nutrition was given in 82.1% of these patients. Identification by Vitek2 v8.1 correlated with MALDI-TOF MS. PCR products of length 163 ​bp were obtained in all isolates as visualized by agarose gel electrophoresis. The biofilm quantity measured as A560 of the twenty-eight C. auris isolates ranged from 0.16 to 0.80 compared to C. albicans. ConclusionsC. auris can be identified by PCR targeting specific rDNA region. Biofilm formation and quantification can be achieved by growing C. auris isolates in Mueller-Hinton broth over a duration of 48 ​h.

Detection of exopolysaccharides (algD, pelF, and pslD) genes in burn wound Pseudomonas aeruginosa isolates

Abstract Background: Pseudomonas aeruginosa biofilm is an essential component of virulence that plays a significant role in antimicrobial resistance and chronic burn wound infections. Objective: This study aimed to investigate the biofilm formation capacity of P. aeruginosa isolated from chronic burn wound from January to May 2022 by biochemical and molecular techniques. Materials and Methods: Quantification of biofilm was performed based on tube method for local isolates of P. aeruginosa after growing on brain heart Broth. The genes encoding exopolysaccharides (algD, pelF, and pslD) were targeted by using conventional polymerase chain reaction (PCR). Results: The results showed that 92.6% of isolates were biofilm former, interestingly 68% of isolates were considered as strong former comparing with other biofilm categories. Gel electrophoresis result of PCR products presented clear bands for algD and psID genes with percentages (96%) and (3.7%) respectively. However, there was no PCR product for pelf gene among all isolates. Conclusion: The prevalence of algD, the large operon necessary for alginate production, was high among P. aeruginosa biofilm producer in this study and it can be an essential agent in the pathogenicity of P. aeruginosa burn wound infections comparing with other biofilm genes (pelF and pslD) of exopolysaccharide structure.

Phenotypic and genotypic characterization of erythromycin-resistant Staphylococcus aureus isolated from bovine subclinical mastitis in Egypt.

Subclinical mastitis (SCM) caused by erythromycin-resistant Staphylococcus aureus is a significant disease in lactating animals. Therefore, it is crucial to understand the genetic factors contributing to erythromycin resistance in S. aureus. This study aimed to estimate the prevalence of S. aureus in milk from subclinical mastitic cattle and buffaloes and tank milk samples as identified by probe-based real-time polymerase chain reaction (PCR) and the genotypic assessment of macrolide and erythromycin resistance profiles, as well as to analyze the phylogenetic relatedness of our local isolates of S. aureus. In total, 285 milk samples were analyzed using the California mastitis test to detect SCM. Milk samples were cultured on different specific Staphylococcus media. The presence of S. aureus was confirmed by Gram staining, the catalase and coagulase tests, the detection of hemolytic activity, DNase agar testing, and biofilm activity in Congo red medium. The genotypic identification of S. aureus (nuc) was performed. The determinants of erythromycin (ermA, ermB, ermC, and ermT) and macrolide resistance (msrA) were screened in all isolates. DNA sequencing of our local isolates of S. aureus was used to analyze their phylogenetic relatedness. Moreover, histopathological examination of tissue specimens of mammary gland was performed. The S. aureus positivity rates were 36.4%, 48.8%, and 63.6% in cattle, buffalo, and bulk tank milk, respectively. Probe-based real-time PCR molecularly confirmed all 62 S. aureus isolates. Thirty-one isolates were subjected to PCR to create profiles of their genotypic erythromycin resistance. ermA, ermB, ermC, and ermT were present in 5 (8%), 26 (41.9%), 18 (29%), and 15 (24.1%) S. aureus isolates, respectively. Moreover, msrA was found in three (4.8%) strains. Eight PCR products were produced using standard PCR for DNA sequencing. Multiple sequence alignment, phylogenetic tree construction, and analysis of nuc in S. aureus revealed a high degree of homology (100%) with S. aureus strains isolated from milk in cases of bovine mastitis in India and Kenya. Histological analysis of udder tissues revealed extensive aggregation of mononuclear inflammatory cells in the interstitial connective tissue, primarily lymphocytes, and macrophages. This study showed a high prevalence of erythromycin resistance in S. aureus isolates. This information is vital for controlling mastitis and the spread of resistance genes between bacterial strains and hosts. Moreover, the probe-based real-time PCR approach is helpful for the rapid screening of S. aureus isolates and the consequent efficient treatment and control of S. aureus mastitis.

Detection of X-Chromosome Inversions in Anopheles Pharoensis Collected from Egypt and Sudan by PCR Assay

Globally, the WHO estimated 241 million malaria cases with 627000 deaths in Africa in 2020. Studying inversions in the X-chromosomes could detect behavioral changes and efficiency of transmission of Malaria in Anopheles phronesis species. These changes may lead to shifting from becoming a major vector of Malaria after the inability to transmit Malaria. The knowledge of the malaria transmission pattern of Anopheles pharoensis in Sudan needs to be improved. We aimed to detect the presence of any inversions or mutations in Anopheles pharoensis species collected from Egypt and Sudan and to undergo a Polymerase Chain Reaction (PCR) technique to detect mutations or X-chromosome inversions in the Sudanese and Egyptian strains of Anopheles pharoensis also to compare between the X-chromosome of the two species. It is an experimental, analytical study that aimed to detect inversions in the X –chromosomes of Anopheles phronesis collected from Egypt (Faiyoum Governate) and Sudan (Khartoum, Gezira, and Sennar states) of Sudanese Mosquito species. From the gel electrophoresis and PCR assay, inversions were detected by the 100Bp PCR products using primers. Only very few revealed bands from 75 Egyptian strains were detected by bands. The study revealed that Anopheles pharoensis species collected from Egypt have X- chromosome inversions. Inversions were absent in the Sudanese strains. The study also reacted to the fact that Anopheles phronesis could be a sibling species or a species complex, but further studies are needed to prove this. From the results and observations of this investigation, it was recommended to undergo further genetic studies on Anopheles species in Sudan and use of PCR technique in the classification.

Molecular Detection of Anaplasma phagocytophilum in Ixodid Ticks Infesting White Fulani Cattle in Zaria and its Environs, Kaduna State, Nigeria.

Anaplasmosis, a disease caused by various species of Anaplasma, poses important economic constraints to animal breeders. In Zaria, molecular detection of tick-borne pathogens infecting cattle is very sketchy. Being hematophagous, ticks are capable of transmitting disease agents such as viruses, bacteria and protozoa. This study was carried out to detect for the presence of Anaplasma pathogen in the various tick species infesting white Fulani breeds of cattle owing to their high population in Zaria and environs using a Polymerase Chain Reaction (PCR). Three hundred and eighty-four (n=384) white Fulani breed of cattle of varying age and sex were sampled from 32 herds in 4 villages (Bomo, Tofu, Ungwan Dabosa and Majeru). The DNA was extracted using Qiagen commercial kit following manufacturer’s instructions. Genomic DNA of the species of ticks was amplified in a semi-nested PCR targeting the 16S rRNA fragment of the Anaplasma spp at expected amplicon size of 711bp. The PCR products were purified using Gel Extraction Kit (Bioland, Scientific LLC) following the manufacturer’s protocol. All sequences were subjected to a Basic Local Alignment Search Tool (BLAST) to determined their identities and assess their homologues and similarities to those in the GenBank. A neighbor-joining tree was generated using the Molecular Engineering Genetic Analysis (MEGA 7.0) to derive the close relationship between the isolates. The result of this study implies that Anaplasma spp (MN044909) is present in all the five species of ticks infesting cattle in Zaria, Kaduna State, Nigeria and pose a high risk on humans and animals health.
 Anaplasmosis, a disease caused by various species of Anaplasma, poses important economic constraints to animal breeders. In Zaria, molecular detection of tick-borne pathogens infecting cattle is very sketchy. Being hematophagous, ticks are capable of transmitting disease agents such as viruses, bacteria and protozoa. This study was carried out to detect for the presence of Anaplasma pathogen in the various tick species infesting white Fulani breeds of cattle owing to their high population in Zaria and environs using a Polymerase Chain Reaction (PCR). Three hundred and eighty-four (n=384) white Fulani breed of cattle of varying age and sex were sampled from 32 herds in 4 villages (Bomo, Tofu, Ungwan Dabosa and Majeru). The DNA was extracted using Qiagen commercial kit following manufacturer’s instructions. Genomic DNA of the species of ticks was amplified in a semi-nested PCR targeting the 16S rRNA fragment of the Anaplasma spp at expected amplicon size of 711bp. The PCR products were purified using Gel Extraction Kit (Bioland, Scientific LLC) following the manufacturer’s protocol. All sequences were subjected to a Basic Local Alignment Search Tool (BLAST) to determined their identities and assess their homologues and similarities to those in the GenBank. A neighbor-joining tree was generated using the Molecular Engineering Genetic Analysis (MEGA 7.0) to derive the close relationship between the isolates. The result of this study implies that Anaplasma spp (MN044909) is present in all the five species of ticks infesting cattle in Zaria, Kaduna State, Nigeria and pose a high risk on humans and animals health.

Bambara Nut Root-Nodules Bacteria from a Semi-Arid Region of South Africa and Their Plant Growth-Promoting Traits.

Rhizobial nitrogen-fixing bacteria are the main inhabitants of the root nodules of legume plants. Studying the bacterial community of legume nodules is important in understanding plant growth and nutrient requirements. Culture-based technique was used to examine the bacterial community of these underground organs from Vigna subterranea L. Verdc (Bambara nut), an underutilized legume in Africa, for plant growth-promoting traits. In this study, Bambara nuts were planted to trap root-nodule bacteria, and the bacteria were morphologically, biochemically, and molecularly characterized. Five selected isolates were screened in vitro for their plant growth-promoting traits and exhibited differences in their phenotypic traits. The polymerase chain reaction (PCR) products were subjected to partial 16S rRNA gene sequencing for phylogenetic analysis. Based on 16S rRNA gene sequence, the isolates were identified as BA1 (Stenotrophomonas maltophilia), BA2 (Chryseobacterium sp.), BA3 (Pseudomonas alcaligenes), BA4 (Pseudomonas plecoglossicida), and BA5 (Pseudomonas hibiscicola). Results showed that four of the five isolates could produce IAA. The capability to solubilize phosphate in Pikovskaya's agar plates was positively shown by four isolates (BA2, BA3, BA4, and BA5). Three isolates could produce hydrogen cyanide while isolates BA1, BA3, BA4, and BA5 were found to have ammonia-production traits. The results suggest that these plant growth-promoting isolates can be used as inoculants for plant growth and productivity.

Molecular Characterization of the NRAMP1 Gene and Blood Parameters of Sinai and Lohman Brown Chickens in Egypt

In almost all animal species, natural resistance-associated macrophage proteins (NRAMPs) have been linked to disease resistance. It plays a crucial part in innate immunity and can affect adaptive immunity as well. The aim of this study was to investigate some immunological traits and molecular genetics in the native breed of chickens, named Sinai (S) and a commercial strain of Lohman Brown (LB). The NRAMP1 gene was reported to be associated with a defense mechanism against infection by bacteria and viruses. A total of 144 female day-old chicks, including 72 from the commercial layer strain (LB) and 72 from the Egyptian native chicken strain (S), were used in this study. At 38 days of age, blood samples were taken randomly from 8 chickens of each group for serum antibodies against the New Castle disease virus, avian influenza virus, and infectious bursal disease virus analysis. Additionally, genomic DNA was extracted from 20 blood samples at 38 days of age. Polymerase chain reaction (PCR) analyses were conducted on the DNA samples, followed by sequencing of the PCR products to identify single nucleotide polymorphisms (SNPs) in the NRAMP1 gene in the two strains of chickens. The findings indicated that lymphocyte, eosinophil, phagocyte activity, and IgY were significantly greater in LB chicks than in S chicks. Sinai chickens, on the other hand, achieved dominance in Newcastle titter. Eight SNPs were found in NRAMP1 of the two strains. The nucleotide identity between S and LB nucleotides was 58.68%, while the changes in different amino acids were found in different positions. Multiple SNPs in the NRAMP1 gene have been discovered in Sinai and LB, suggesting that this gene can be used as a genetic marker for the selection of high-producing indigenous hybrids with the ability to resist pathogenic diseases in poultry.

Phenotypic and Molecular Investigation of Streptococcus pneumoniae pneumolysin

The current study aimed to detect the prevalence of Streptococcus pneumoniae by identifying Pneumolysin and determining the gene (ply) using polymerase chain reaction (PCR). The study aimed to highlight the isolation and identification of Streptococcus pneumoniae using morphological, biochemical and Vitek, as well as investigation about pneumolysin phenotypically and molecularly through ply gene and sent the PCR products to sequencing by sanger method. Fifty sputum specimens were collected from patients at AL Salam Hospital, Iben Sina/ Mosul/ Iraq, from August 2021 to March 2022. The isolated bacteria were identified depending on morphology and biochemical properties; Vitek and the ply gene were detected by PCR technique. Five isolates of Streptococcus pneumoniae showed the ability to produce pneumolysin when tested by a double agar layer. When PCR reaction was performed on the Streptococcus pneumoniae pneumolysin gene, the results on gel electrophoresis showed three bands with 238 bp, and the ratio of the presence of the ply gene was 80%. PCR products were then submitted to sequencing by the Sanger method, and the ply sequencing result showed Point mutations that nucleotide and amino acid change with location. Keywords: Streptococcus pneumonia; pneumolysin; ply gene; PCR sequencing.

Central nervous system mycobacteriosis caused by Mycobacterium genavense in degus (Octodon degus).

Degus (Octodon degus) that were kept at a breeding facility presented with neurological or respiratory symptoms and died. Necropsies were performed on 9 individuals, and no significant gross lesions were found. Histologically, spinal cord necrosis was observed in all 9 cases and granulomatous myelitis in 5 of the 9 cases. Locally extensive necrosis of the brain and encephalitis were observed in 7 of the 9 cases. Acid-fast bacteria were found in the spinal cords, brains, and lungs from all 9 cases. Immunohistochemically, Mycobacterium tuberculosis antigen was observed in the spinal cords, brains, and lungs from all 9 cases. Double-labeling immunofluorescence revealed M. tuberculosis antigen in IBA1- and myeloperoxidase-immunopositive cells. Extracted genomic DNA from 8 of the 9 cases was successfully amplified with the primers for Mycobacterium genavense ITS1 and hypothetical 21 kDa protein genes, and the polymerase chain reaction products were identified as M. genavense by DNA sequencing. This report highlights the susceptibility of degus to M. genavense infection in the central nervous system.

Molecular phylogeny and morphological studies reveal a 30‐year‐old rain tree (Samanea saman) maintains populations of Meloidogyne enterolobii, a new host plant in Thailand

AbstractSamanea saman (Jacq.) Merr., commonly known as rain tree, is a crucial shade tree with traditional medicinal properties. In our study, 50 soil samples were collected around the rhizosphere of rain tree in Bangkok, Pathum Thani, Uttaradit, Khon Kaen and Ubon Ratchathani Provinces in Thailand. Meloidogyne populations were only found in soil samples from Mueang district, Khon Kaen Province and a 30‐year‐old rain tree exhibited root gall symptoms caused by root‐knot nematodes (Meloidogyne) on its adventitious roots. The species of the nematodes were identified based on its morphological and molecular characteristics. The results demonstrated that the perineal patterns and morphometrics of the nematodes were highly similar to the original description of M. enterolobii, except for a squarish dorsal arch and two faint lateral lines. During the molecular analysis, nematode DNA was extracted and amplified using three primer sets, C2F3/1108, D2A/D3B and MK7F/MK7R SCAR primer, to generate polymerase chain reaction (PCR) products of 700, 750 and 520 bp, respectively. The polygenetic tree of the two markers displayed similarity among the COII and the 16S rRNA gene in mitochondria and the D2/D3 region in 28S rRNA. Therefore, the nematodes were identified as M. enterolobii Yang and Eisenback, 1983. This study reports a new host record of M. enterolobii on rain tree.